Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that, despite being treatable with well- defined chemotherapy regimens, is ultimately fatal in over half of all cases categorized as high-risk AML. Mutations in MLL, FLT3, DNMT3A and P53 are associated with high-risk AML. Even targeted FLT3 anti kinase therapy, which constitutes 30 % of AML, failed to engender durable response in this group of AML. Co-operative oncogenic signaling? was attributed to poor therapeutic outcome, but lacks mechanistic understanding. Based on our recent publication and new preliminary data, we found that co-operative oncogenic signaling converges on c-FOS and DUSP1, which results in an increased apoptotic threshold in cancer cells and confers drug resistance. Thus, genetic or pharmacologic inhibition of c-FOS and DUSP1 sensitizes cancer cells to chemotherapy (Kesarwani, et. al. Nature Medicine 2017). We show greater expression of c-FOS and DUSP1 in high-risk AML patients, but not in low risk-AML patients. Both genetic and chemical inhibition of c-FOS and DUSP1 results in increased drug sensitivity to both TKI and conventional chemotherapeutic drugs in a model of high-risk AML (FLT3ITD+MLLAF9). Thus, we hypothesize that co-operative oncogenic signaling in AML induces the expression of c-FOS and DUSP1 resulting to drug resistance and disease relapse due to elevated apoptotic threshold. In Aim 1, we will determine whether c-FOS and DUSP1 are necessary and sufficient for transformation in a most frequent, aggressive, and fatal AML driven by FLT3ITD+DNMT3Amut+NPM1C and FLT3ITD+P53mut mutations. We will examine the cellular basis of c-FOS and DUSP1 dependency in the high- risk AML mouse models and primary patient samples by genetic deletion and pharmacological inhibition of c- FOS and DUSP1. Next, we propose experiments to understand the mechanistic basis for how co-operative oncogenic signaling via c-FOS and DUSP1 contributes to transformation and treatment failure in AML, with the goal for novel treatment strategies. Based on our preliminary data, we hypothesize that c-FOS and DUSP1 signaling converges upon oncogenically-activated enhancers mediated by specific AP-1 transcriptional complexes. In the presence of c-FOS and DUSP1, AP-1 complexes consist of c-FOS-JUN, which mediate oncogenically-active enhancers, while in the absence of c-FOS and DUSP1, Jun family homodimers (JUN- JUNB, JUNB-JUND, JUN-JUND) predominate which are unable to support the leukemic cell state. In Aim 2, we will molecularly link c-FOS-JUN AP-1 and DUSP1 activity to global enhancer chromatin dynamics. Moreover, we will exploit chromatin-embedded target-gene-reporter alleles to provide a detailed analysis of functionally-relevant downstream genes at a single-cell level in high-risk AML. The proposed work is expected to delineate the necessity of c-FOS and DUSP1 signaling in high-risk AML, as well as to provide deep molecular insight into the mechanisms underlying leukemic transformation and drug resistance. We expect that this information will be informative not only for AML, but also the broad group of treatment resistant tumors.